The present invention is related to guanine nucleotide-binding proteins. More particularly, the present invention is related to specific reagents and probes for identifying, isolating and distinguishing between different guanine nucleotide (GTP)-binding proteins.
State of The Art:
GTP-binding (hereinafter "G" proteins) comprise a family of distinct but related signal transducers. One or more members of the family is found in virtually every type of cell, and performs a critical role in the transduction of hormonal, neurotransmitter, cytokine, odorant, and light signal functions. G-proteins are known to be heteromeric; the alpha-GTP-binding subunit is distinct for each member of the G-protein family and is believed to confer specificity in both receptor and effector interactions.
A combination of protein purification and recombinant DNA techniques have led to the realization that the G-protein family consists of at least 7 distinct members. Cloning of complementary DNA for each of these allows prediction of the amino acid sequence. The proteins include 2 forms of "transducin" (TD), a G-protein uniquely found in retinal photoreceptors and involved in phototransduction, and five other proteins termed G.sub.s, G.sub.o, G.sub.il, G.sub.i2, and G.sub.i3. The specific functions of each of these is as yet unclear, but they appear to regulate ion channels and enzymes that generate intercellular "second messengers." Although each protein shows significant amino acid sequence homology (ranging from 40% for G.sub.s vs. G.sub.i, to 90% for G.sub.il vs. G.sub.i3), there are unique sequence differences in each.
Certain bacterial toxins, particularly pertussis toxin, covalently modify certain G-proteins and this technique has been used for identification of G-proteins. However, this method is relatively nonspecific as transducin (TD), G.sub.o, and all forms of G.sub.i appear to be pertussis toxin substrates. For this reason, several laboratories have generated antibodies against G-proteins to obtain more specific probes. The majority of these reagents, whether polyclonal rabbit antisera or monoclonal mouse antibodies are of undefined epitope, and are not particularly specific for a single G-protein so as to definitively distinguish one from the other. Moreover, none of the known antibodies can be used to block specific functional interactions of G-proteins.